History and Prospects of Drug Discovery and Development Collaboration between Industry and Academia.

Research collaborations and licensing deals are critical for the discovery and development of life-saving drugs. This practice has been ongoing since the inception of the pharmaceutical industry. The current process of drug discovery and development is complex, regulated, and highly regimented, having evolved over time. Academia excels in the discovery of fundamental scientific concepts and biological processes, while industry excels in translational science and product development. Potential for collaboration exists at every step of the drug discovery and development continuum. This perspective walks through such collaborative activities, provides examples, and offers tips for potential collaborations.

DFT investigation of coupling constant anomalies in substituted ß-lactams.

ß-lactams are a chemically diverse group of molecules with a wide range of biological activities. Having recently observed curious trends in 2JHH coupling values in studies on this structural class, we sought to obtain a more comprehensive understanding of these diagnostic NMR parameters, specifically interrogating 1JCH, 2JCH, and 2JHH, to differentiate 3- and 4-monosubstituted ß-lactams. Further investigation using computational chemistry methods was employed to explore the geometric and electronic origins for the observed and calculated differences between the two substitution patterns.

Computational studies of [1]benzothieno[2,3-c]naphtho[1,2-f]quinoline.

Early NMR studies of several heterohelicenes containing an annular nitrogen atom and a thiophene ring in their structure suggested the possibility of the lengthening of the carbon-carbon bonds in the interior of the helical turn of the molecule based on the progressive more shielded nature of 13 C resonances toward the center of the helical turn. Computational chemistry capabilities when those NMR studies were performed were primitive in comparison to what is now possible. We now report the optimized geometry and a comparison of the calculated versus observed 1 H and 13 C NMR chemical shift assignments for [1]benzothieno[2,3-c]naphtho[1,2-f]quinoline that confirms these suspicions.

J-modulated 19 F- and 1 H-detected dual-optimized inverted 1 JCC 1,n-ADEQUATE: A universal ADEQUATE experiment.

The recently reported 19 F-detected dual-optimized inverted 1 JCC 1,n-ADEQUATE experiment and the previously reported 1 H-detected version have been modified to incorporate J-modulation, making it feasible to acquire all 1,1- and 1,n-ADEQUATE correlations as well as 1 JCC and n JCC homonuclear scalar couplings in a single experiment. The experiments are demonstrated using N,N-dimethylamino-2,5,6-trifluoro-3,4-phthalonitrile and N,N-dimethylamino-3,4-phthalonitrile.

DELTA50: A Highly Accurate Database of Experimental 1H and 13C NMR Chemical Shifts Applied to DFT Benchmarking.

Density functional theory (DFT) benchmark studies of 1H and 13C NMR chemical shifts often yield differing conclusions, likely due to non-optimal test molecules and non-standardized data acquisition. To address this issue, we carefully selected and measured 1H and 13C NMR chemical shifts for 50 structurally diverse small organic molecules containing atoms from only the first two rows of the periodic table. Our NMR dataset, DELTA50, was used to calculate linear scaling factors and to evaluate the accuracy of 73 density functionals, 40 basis sets, 3 solvent models, and 3 gauge-referencing schemes. The best performing DFT methodologies for 1H and 13C NMR chemical shift predictions were WP04/6-311++G(2d,p) and ωB97X-D/def2-SVP, respectively, when combined with the polarizable continuum solvent model (PCM) and gauge-independent atomic orbital (GIAO) method. Geometries should be optimized at the B3LYP-D3/6-311G(d,p) level including the PCM solvent model for the best accuracy. Predictions of 20 organic compounds and natural products from a separate probe set had root-mean-square deviations (RMSD) of 0.07 to 0.19 for 1H and 0.5 to 2.9 for 13C. Maximum deviations were less than 0.5 and 6.5 ppm for 1H and 13C, respectively.

Biodistribution and Activity of EGFR Targeted Polymeric Micelles Delivering a New Inhibitor of DNA Repair to Orthotopic Colorectal Cancer Xenografts with Metastasis.

The disruption of polynucleotide kinase/phosphatase (PNKP) in colorectal cancer (CRC) cells deficient in phosphatase and tensin homolog (PTEN) is expected to lead to the loss of cell viability by a process known as synthetic lethality. In previous studies, we have reported on the encapsulation of a novel inhibitor of PNKP, namely, A83B4C63, in polymeric micelles and its activity in slowing the growth of PTEN-deficient CRC cells as well as subcutaneous xenografts. In this study, to enhance drug delivery and specificity to CRC tumors, the surface of polymeric micelles carrying A83B4C63 was modified with GE11, a peptide targeting epidermal growth factor receptor (EGFR) overexpressed in about 70% of CRC tumors. Using molecular dynamics (MD) simulations, we assessed the binding site and affinity of GE11 for EGFR. The GE11-modified micelles, tagged with a near-infrared fluorophore, showed enhanced internalization by EGFR-overexpressing CRC cells in vitro and a trend toward increased primary tumor homing in an orthotopic CRC xenograft in vivo. In line with these observations, the GE11 modification of polymeric micelles was shown to positively contribute to the improved therapeutic activity of encapsulated A83B4C63 against HCT116-PTEN-/- cells in vitro and that of orthotopic CRC xenograft in vivo. In conclusion, our results provided proof of principle evidence for the potential benefit of EGFR targeted polymeric micellar formulations of A83B4C63 as monotherapeutics for aggressive and metastatic CRC tumors but at the same time highlighted the need for the development of EGFR ligands with improved physiological stability and EGFR binding.

One-Step Regio- and Stereoselective Electrochemical Synthesis of Orexin Receptor Antagonist Oxidative Metabolites.

Synthesis of drug metabolites, which often have complex structures, is an integral step in the evaluation of drug candidate metabolism, pharmacokinetic (PK) properties, and safety profiles. Frequently, such synthetic endeavors entail arduous, multiple-step de novo synthetic routes. Herein, we present the one-step Shono-type electrochemical synthesis of milligrams of chiral α-hydroxyl amide metabolites of two orexin receptor antagonists, MK-8133 and MK-6096, as revealed by a small-scale (pico- to nano-mole level) reaction screening using a lab-built online electrochemistry (EC)/mass spectrometry (MS) (EC/MS) platform. The electrochemical oxidation of MK-8133 and MK-6096 was conducted in aqueous media and found to produce the corresponding α-piperidinols with exclusive regio- and stereoselectivity, as confirmed by high-resolution nuclear magnetic resonance (NMR) characterization of products. Based on density functional theory (DFT) calculations, the exceptional regio- and stereoselectivity for this electrochemical oxidation are governed by more favorable energetics of the transition state, leading to the preferred secondary carbon radical α to the amide group and subsequent steric hindrance associated with the U-shaped conformation of the cation derived from the secondary α-carbon radical, respectively.

Prediction of anisotropic NMR data without knowledge of alignment medium structure by surface decomposition.

Prediction of anisotropic NMR data directly from solute-medium interaction is of significant theoretical and practical interest, particularly for structure elucidation, configurational analysis and conformational studies of complex organic molecules and natural products. Current prediction methods require an explicit structural model of the alignment medium: a requirement either impossible or impractical on a scale necessary for small organic molecules. Here we formulate a comprehensive mathematical framework for a parametrization protocol that deconvolutes an arbitrary surface of the medium into several simple local landscapes that are distributed over the medium's surface by specific orientational order parameters. The shapes and order parameters of these local landscapes are determined via fitting that maximizes the congruence between experimentally determined anisotropic NMR measurables and their predicted counterparts, thus avoiding the need for an a priori knowledge of the global medium morphology. This method achieves substantial improvements in the accuracy of predicted anisotropic NMR values compared to current methods, as demonstrated herein with sixteen natural products. Furthermore, because this formalism extracts structural commonalities of the medium by combining anisotropic NMR data from different compounds, its robustness and accuracy are expected to improve as more experimental data become available for further re-optimization of fitting parameters.

Application of 1,1-ADEQUATE and DFT to correct 13 C misassignments of carbonyl chemical shifts for carbapenem antibiotics.

Prior to the development of sensitive proton-detected 2D NMR experiments, assigning 13 C signals could be a significant challenge, and mistakes have occurred even for prominent compound classes. In this study, 1,1-ADEQUATE data were used to unambiguously reassign the 13 C chemical shifts for the ß-lactam carbonyl at the C-7 position and the proximal carboxylate at the C-10 position of the carbapenems, meropenem and imipenem. Density functional theory (DFT) was then investigated to provide sufficiently accurate 13 C chemical shift predictions, allowing for the carbonyl signal reassignment of thienamycin.

19 F-detected dual-optimized inverted 1 JCC 1,n-ADEQUATE.

Modification of the recently reported 19 F-detected 1,1-ADEQUATE experiment that incorporates dual-optimization to selectively invert a wide range of 1 JCC correlations in a 1,n-ADEQUATE experiment is reported. Parameters for the dual-optimization segment of the pulse sequence were modified to accommodate the increased size of 1 JCC homonuclear coupling constants of poly- and perfluorinated molecules relative to protonated molecules to allow broadband inversion of the 1 JCC correlations. The observation and utility of isotope shifts are reported for the first time for 1,1- and 1,n-ADEQUATE correlations.

Development of 19 F-detected 1,1-ADEQUATE for the characterization of polyfluorinated and perfluorinated compounds.

Polyfluorinated and perfluorinated compounds in the environment are a growing health concern. 19 F-detected variants of commonly employed heteronuclear shift correlation experiments such as heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) are available; 19 F-detected experiments that employ carbon-carbon homonuclear coupling, in contrast, have never been reported. Herein, we report the measurement of the 1 JCC and n JCC coupling constants of a simple perfluorinated phthalonitrile and the first demonstration of a 19 F-detected 1,1-ADEQUATE experiment.

Repurposing Melt Degradation for the Evaluation of Mixed Amorphous-Crystalline Blends.

Medicine regulators require the melting points for crystalline drugs, as they are a test for chemical and physical quality. Many drugs, especially salt-forms, suffer concomitant degradation during melting; thus, it would be useful to know if the endotherm associated with melt degradation may be used for characterising the crystallinity of a powder blend. Therefore, the aim of this study was to investigate whether melt-degradation transitions can detect amorphous content in a blend of crystalline and amorphous salbutamol sulphate. Salbutamol sulphate was rendered amorphous by freeze and spray-drying and blended with crystalline drug, forming standards with a range of amorphous content. Crystalline salbutamol sulphate was observed to have a melt-degradation onset of 198.2±0.2°C, while anhydrous amorphous salbutamol sulphate prepared by either method showed similar glass transition temperatures of 119.4±0.7°C combined. Without the energy barrier provided by the ordered crystal lattice, the degradation endotherm for amorphous salbutamol sulphate occurred 50°C below the melting point, with an onset of 143.6±0.2°C. The enthalpies for this degradation transition showed no significant difference between freeze- and spray-dried samples (p>0.05). Distinct from convention, partial integration of the crystalline melt-degradation endotherm was applied to the region 193-221°C which had no contribution from the degradation of amorphous salbutamol sulphate. The linear correlation of these partial areas with amorphous content, R2=0.994, yielded limits of detection and quantification of 0.13% and 0.44% respectively, independent of drying technique. Melt-degradation transitions may be re-purposed for the measurement of amorphous content in powder blends, and they have potential for evaluating disorder more generally.

Probing the Molecular-Level Interactions in an Active Pharmaceutical Ingredient (API) - Polymer Dispersion and the Resulting Impact on Drug Product Formulation.

PURPOSE: An investigation of underlying mechanisms of API-polymer interaction patterns has the potential to provide valuable insights for selecting appropriate formulations with superior physical stability and processability. MATERIALS AND METHODS: In this study, copovidone was used as a polymeric carrier for several model compounds including clotrimazole, nifedipine, and posaconazole. The varied chemical structures conferred the ability for the model compounds to form distinct interactions with copovidone. Rheology and nuclear magnetic resonance (NMR) were combined to investigate the molecular pattern and relative strength of active pharmaceutical ingredient (API)-polymer interactions. In addition, the impact of the interactions on formulation processability via hot melt extrusion (HME) and physical stability were evaluated. RESULTS: The rheological response of an API-polymer system was found to be highly sensitive to API-polymer interaction, depending both on API chemistry and API-polymer miscibility. In the systems studied, dispersed API induced a stronger plasticizer effect on the polymer matrix compared to crystalline/aggregated API. Correspondingly, the processing torque via HME showed a proportional relationship with the maximum complex viscosity of the API-polymer system. In order to quantitatively evaluate the relative strength of the API-polymer interaction, homogeneously dispersed API-polymer amorphous samples were prepared by HME at an elevated temperature. DSC, XRD, and rheology were employed to confirm the amorphous integrity and homogeneity of the resultant extrudates. Subsequently, the homogeneously dispersed API-polymer amorphous dispersions were interrogated by rheology and NMR to provide a qualitative and quantitative assessment of the nature of the API-polymer interaction, both macroscopically and microscopically. Rheological master curves of frequency sweeps of the extrudates exhibited a strong dependence on the API chemistry and revealed a rank ordering of the relative strength of API-copovidone interactions, in the order of posaconazole > nifedipine > clotrimazole. NMR data provided the means to precisely map the API-polymer interaction pattern and identify the specific sites of interaction from a molecular perspective. Finally, the impact of API-polymer interactions on the physical stability of the resultant extrudates was studied. CONCLUSION: Qualitative and quantitative evaluation of the relative strength of the API-polymer interaction was successfully accomplished by utilizing combined rheology and NMR. Graphical Abstract.

Developing nonuniform sampling strategies to improve sensitivity and resolution in 1,1-ADEQUATE experiments.

Nonuniform sampling (NUS) strategies are developed for acquiring highly resolved 1,1-ADEQUATE spectra, in both conventional and homodecoupled (HD) variants with improved sensitivity. Specifically, the quantile-directed and Poisson gap methods were critically compared for distributing the samples nonuniformly, and the quantile schedules were further optimized for weighting. Both maximum entropy and iterative soft thresholding spectral estimation algorithms were evaluated. All NUS approaches were robust when the degree of data reduction is moderate, on the order of a 50% reduction of sampling points. Further sampling reduction by NUS is facilitated by using weighted schedules designed by the quantile method, which also suppresses sampling noise well. Seed independence and the ability to specify the sample weighting in quantile scheduling are important in optimizing NUS for 1,1-ADEQUATE data acquisition. Using NUS yields an improvement in sensitivity, while also making longer evolution times accessible that would be difficult or impractical to attain by uniform sampling. Theoretical predictions for the sensitivity enhancements in these experiments are in the range of 5-20%; NUS is shown to disambiguate weak signals, reveal some n JCC correlations obscured by noise, and improve signal strength relative to uniform sampling in the same experimental time. This work presents sample schedule development for applying NUS to challenging experiments. The schedules developed here are made available for general use and should facilitate the broader utilization of ADEQUATE experiments (including 1,1-, 1,n-, and HD- variants) for challenging structure elucidation problems.

Longitudinal analysis of communication repair skills across three neurodevelopmental disabilities.

BACKGROUND: This study is a longitudinal follow-up to prior work examining the important pragmatic skill of communication repair (i.e., the ability to respond effectively to a request for clarification of an unclear message) across three neurodevelopmental disabilities in which language skills are impaired: fragile X syndrome with and without autism spectrum disorder (FXS-ASD; FXS-O), idiopathic ASD (ASD-O), Down syndrome (DS) and controls with typical development (TD). Prior work examining communication repair skills at younger ages indicated impairments in boys with FXS-ASD and ASD-O, with females performing comparably with each other across groups. AIMS: To characterize communication repair skills in young individuals with FXS-ASD, FXS-O, ASD-O, DS and TD, across groups and over development. A secondary aim included documenting sex differences in FXS (with and without ASD) and DS. METHODS & PROCEDURES: Sixty young individuals with FXS-ASD (49 males, 11 females), 38 with FXS-O (13 males, 25 females), 38 with ASD-O (males only), 42 with DS (21 males, 21 females) and 41 with TD (21 males, 20 females) participated in the study, with a subsample reported on here who were retested at a second time point 2.7 years later on average. Participants completed a structured, picture-based task designed to assess the ability to repair breakdowns in communication. Participants' responses were compared across groups and sexes at the second time point, and interpreted with respect to previously published (Time 1) findings. OUTCOMES & RESULTS: Key findings included that, with age, male groups (including those with FXS-ASD and ASD-O, who showed difficulty at Time 1) performed more comparably, decreasing their use of inappropriate responses, in spite of relatively little change observed in general cognitive or structural language abilities in the clinical groups. However, girls with FXS and DS became more non-responsive with age, and differences between boys and girls with FXS-ASD emerged over time as well. CONCLUSIONS & IMPLICATIONS: Findings suggest that impairments in a critical pragmatic skill-the ability to repair communication breakdown-show significant change with age across three neurodevelopmental disabilities, with important sex-specific patterns. These developments were often observed in spite of a relative plateau in cognitive and language growth, suggesting that repair skills may be more malleable and therefore an excellent target for intervention. Findings not only inform the nature of pragmatic impairment across groups but also can importantly inform clinical practice, suggesting that clinicians should monitor pragmatic skills such as repairs throughout development and also consider the role of sex in clinical efforts.

Determination of Complex Small-Molecule Structures Using Molecular Alignment Simulation.

Correct structural assignment of small molecules and natural products is critical for drug discovery and organic chemistry. Anisotropy-based NMR spectroscopy is a powerful tool for the structural assignment of organic molecules, but it relies on the utilization of a medium that disrupts the isotropic motion of molecules in organic solvents. Here, we establish a quantitative correlation between the atomic structure of the alignment medium, the molecular structure of the small molecule, and molecule-specific anisotropic NMR parameters. The quantitative correlation uses an accurate three-dimensional molecular alignment model that predicts residual dipolar couplings of small molecules aligned by poly(γ-benzyl-l-glutamate). The technique facilitates reliable determination of the correct stereoisomer and enables unequivocal, rapid determination of complex molecular structures from extremely sparse NMR data.

Unexpected Propargylic Retro-Brook Rearrangements in Alkynes.

Retro-Brook rearrangements refer to the intramolecular migration of a silyl group from oxygen to carbon. In this study, we report a novel propargylic retro-Brook rearrangement observed in terminal alkynes bearing a silyl ether moiety. Retro-Brook rearrangements involving [1,2]-, [1,4]-, and [1,5]-migrations are described, affording propargylsilanes in reasonable yield. The reaction mechanism was investigated experimentally by deuterium quenching and rationalized by density functional theory calculations. The terminal alkyne and the subsequent propargyl/allenyl dianion were shown to be crucial for the reaction favoring the retro-Brook rearrangement product over the Brook rearrangement. The second deprotonation at the propargylic position was determined to be the rate-limiting step. In addition, a gas-phase Brook-type rearrangement of the propargylsilanes was observed under GC-MS conditions. This observation was also further confirmed by DFT calculations.

Microbial biotransformation - an important tool for the study of drug metabolism.

Metabolite identification is an integral part of both preclinical and clinical drug discovery and development. Synthesis of drug metabolites is often required to support definitive identification, preclinical safety studies and clinical trials. Here we describe the use of microbial biotransformation as a tool to produce drug metabolites, complementing traditional chemical synthesis and other biosynthetic methods such as hepatocytes, liver microsomes and recombinant human drug metabolizing enzymes. A workflow is discussed whereby microbial strains are initially screened for their ability to form the putative metabolites of interest, followed by a scale-up to afford quantities sufficient to perform definitive identification and further studies. Examples of the microbial synthesis of several difficult-to-synthesize hydroxylated metabolites and three difficult-to-synthesize glucuronidated metabolites are described, and the use of microbial biotransformation in drug discovery and development is discussed.

CyClick Chemistry for the Synthesis of Cyclic Peptides.

Here, we report a novel "CyClick" strategy for the macrocyclization of peptides that works in an exclusively intramolecular fashion thereby precluding the formation of dimers and oligomers via intermolecular reactions. The CyClick chemistry is highly chemoselective for the N-terminus of the peptide with a C-terminal aldehyde. In this protocol, the peptide conformation internally directs activation of the backbone amide bond and thereby facilitates formation of a stable 4-imidazolidinone-fused cyclic peptide with high diastereoselectivity (>99 %). This method is tolerant to a variety of peptide aldehydes and has been applied for the synthesis of 12- to 23-membered rings with varying amino acid compositions in one pot under mild reaction conditions. The reaction generated peptide macrocycles featuring a 4-imidazolidinone in their scaffolds, which acts as an endocyclic control element that promotes intramolecular hydrogen bonding and leads to macrocycles with conformationally rigid turn structures.

A Randomized Trial on the Efficacy of Mastery Learning for Primary Care Provider Melanoma Opportunistic Screening Skills and Practice.

BACKGROUND: Early detection of melanoma represents an opportunity to reduce the burden of disease among people at increased risk for melanoma. OBJECTIVE: To develop and demonstrate the efficacy of online training. DESIGN: Randomized educational trial. PARTICIPANTS: Primary care providers (PCPs). INTERVENTION: Mastery learning course with visual and dermoscopic assessment, diagnosis and management, and deliberate practice with feedback to reach a minimum passing standard. MAIN MEASURES: Pre-test/post-test diagnostic accuracy. Referral of concerning lesions for 3 months before and after the educational intervention. KEY RESULTS: Among the 89 PCPs, 89.8% were internal medicine physicians, and the remainder were physician assistants embedded in internists' practices. There were no differences between control and intervention groups regarding gender, age, race, or percentage of full-time PCPs. The control group had more PCPs who reported less than 5 years of practice (n = 18) than the intervention group (n = 6) (χ2 [6, n = 89] = 14.34, p = 0.03). PCPs in the intervention group answered more melanoma detection questions correctly on the post-test (M = 10.05, SE = 1.24) compared to control group PCPs (M = 7.11, SE = 0.24), and had fewer false-positive and no false-negative melanoma diagnoses (intervention, M = 1.09, SE = 0. 20; control, M = 3.1, SE = 0.23; ANCOVA, F[1,378] =27.86, p < 0.001; ηp2 = 0.26). PCPs who underwent training referred fewer benign lesions, including nevi, seborrheic keratoses, and dermatofibromas, than control PCPs (F[1,79] = 72.89, p < 0.001; ηp2 = 0.489; F[1,79] = 25.82, p < 0.001; ηp2 = 0.246; F[1,79] = 34.25, p < 0.001; ηp2 = 0.302; respectively). Those receiving training referred significantly more melanomas than controls (F[1,79] = 24.38, p < 0.001; ηp2 = 0.236). Referred melanomas (0.8 ± 0.07 per month for intervention, 0.17 ± 0.06 for control) were mostly located on the head and neck. CONCLUSIONS: Mastery learning improved PCPs' ability to detect melanoma on a standardized post-test and may improve referral of patients with suspected melanoma. Further studies are needed to confirm this finding. ClinicalTrials.gov NCT02385253.